Lepidopteran GABA gated chloride channel and nucleic acids encoding subunits thereof

The present invention provides isolated nucleic acids encoding lepidopteran gamma aminobutyric acid (GABA) gated chloride channels. In a preferred embodiment, the nucleic acids encoding lepidopteran GABA gated chloride channels are isolatable from Heliothis virescens. The invention further provides recombinant lepidopteran GABA gated chloride channels and methods for identifying agonists and antagonists to a lepidopteran GABA gated chloride channels.

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Claims

1. An isolated nucleic acid encoding a lepidopteran GABA gated chloride channel subunit comprising the sequence of at least one of SEQ ID NO: 1, SEQ ID NO: 3, nucleotides 115 to 1581 of SEQ ID NO: 1, nucleotides 115 to 1581 of SEQ ID NO: 3, a nucleic acid encoding the polypeptide of SEQ ID NO:2 or a nucleic acid encoding the polypeptide of SEQ ID NO:4.

2. An isolated nucleic acid encoding a lepidopteran GABA gated chloride channel subunit capable of hybridizing at about 50.degree. C., 2x SSC to at least one of the complement of SEQ ID NO: 1, the complement of SEQ ID NO: 3, the complement of a nucleic acid encoding the polypeptide of SEQ ID NO: 2 or the complement of a nucleic acid encoding the polypeptide of SEQ ID NO:4.

3. An isolated nucleic acid having a sequence encoding the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

4. An expression vector comprising the isolated nucleic acid of any one of claims 1, 2, or 3.

5. A host cell comprising an expression vector comprising the isolated nucleic acid of claim 1, 3 or 5.

6. The host cell of claim 5 wherein said host cell is a bacterial cell, a yeast cell, an insect cell or a mammalian cell.

7. A Xenopus oocyte comprising the isolated nucleic acid of claim 1, 2 or 3.

8. Recombinant lepidopteran GABA gated chloride channel comprising the amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 4.

9. A Xenopus oocyte expressing at least one functional lepidopteran GABA gated chloride channel comprising the amino acid sequence as set forth in at least one of SEQ ID NO: 2 or SEQ ID NO: 4.

10. An insect cell expressing at least one functional recombinant lepidopteran GABA gated chloride channel comprising the amino acid sequence as set forth in at least one of SEQ ID NO: 2 or SEQ ID NO: 4.

11. The cell of claim 10 wherein said cell is an Sf9 cell.

12. A method for preparing a lepidopteran GABA gated chloride channel comprising culturing a host cell comprising the expression vector of claim 4 under conditions suitable for expression.

13. A method for preparing a Xenopus oocyte expressing at least one functional lepidopteran GABA gated chloride channel which comprises injecting a Xenopus oocyte with a nucleic acid encoding, at least one lepidopteran GABA gated chloride channel, wherein the nucleic acid is selected from the group consisting of a nucleic acid having the sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or nucleotides 115 to 1581 of SEQ ID NO: 1 or nucleotides 115 to 1581 of SEQ ID NO: 3, a nucleic acid encoding the polypeptide of SEQ ID NO:2, a nucleic acid encoding the polypeptide of SEQ ID NO:4, a nucleic acid capable of hybridizing at about 50.degree. C., 2x SSC, to the complement of a nucleic acid having SEQ ID NO: 1, a nucleic acid capable of hybridizing at about 50.degree. C., 2x SSC, to the complement of a nucleic acid having SEQ ID NO: 3, a nucleic acid capable of hybridizing at about 50.degree. C., 2X SSC, to the complement of a nucleic acid encoding the polypeptide of SEQ ID NO: 2, and a nucleic acid capable of hybridizing at about 50.degree. C., 2X SSC, to the complement of a nucleic acid encoding the polypeptide of SEQ ID NO: 4 and culturing said oocyte under conditions suitable for expression of said lepidopteran GABA gated chloride channel.

14. A composition comprising a eukaryotic cell membrane containing at least one functional lepidopteran GABA gated chloride channel, encoded by a nucleic acid selected from the group consisting of a nucleic acid having the sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or nucleotides 115 to 1581 of SEQ ID NO: 1 or nucleotides 115 to 1581 of SEQ ID NO: 3, a nucleic acid encoding the polypeptide of SEQ ID NO:2, a nucleic acid encoding the polypeptide of SEQ ID NO:4, a nucleic acid capable of hybridizing at about 50.degree. C., 2x SSC, to the complement of a nucleic acid having SEQ ID NO: 1, a nucleic acid capable of hybridizing at about 50.degree. C., 2x SSC, to the complement of a nucleic acid having SEQ ID NO: 3, a nucleic acid capable of hybridizing at about 50.degree. C., 2X SSC, to the complement of a nucleic acid encoding the polypeptide of SEQ ID NO: 2, and a nucleic acid capable of hybridizing at about 50.degree. C., 2X SSC, to the complement of a nucleic acid encoding the polypeptide of SEQ ID NO: 4.

15. A composition comprising a eukaryotic host cell expressing a functional lepidopteran GABA gated chloride channel, encoded by a nucleic acid selected from the group consisting of a nucleic acid having the sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or nucleotides 115 to 1581 of SEQ ID NO: 1 or nucleotides 115 to 1581 of SEQ ID NO: 3, a nucleic acid encoding the peptide of SEQ ID NO:2, a nucleic acid encoding the peptide of SEQ ID NO:4, a nucleic acid capable of hybridizing at about 50.degree. C., 2x SSC, to the complement of a nucleic acid having SEQ ID NO: 1, a nucleic acid capable of hybridizing at about 50.degree. C., 2x SSC, to the complement of a nucleic acid having SEQ ID NO: 3, a nucleic acid capable of hybridizing at about 50.degree. C., 2X SSC, to the complement of a nucleic acid encoding the polypeptide of SEQ ID NO: 2, and a nucleic acid capable of hybridizing at about 50.degree. C., 2X SSC, to the complement of a nucleic acid encoding the polypeptide of SEQ ID NO: 4.

16. A kit containing a first container containing a recombinant lepidopteran GABA gated chloride channel according to claim 8 in a cell membrane.

17. The kit of claim 16 wherein said cell membrane is a membrane preparation, a freeze dried membrane preparation, an intact cell or an oocyte.

Referenced Cited
U.S. Patent Documents
5487976 January 30, 1996 Soderlund et al.
Foreign Patent Documents
93/07161 April 1993 WOX
Other references
  • Anthony et al., "GABA receptor molecules of insects", in Comparative Molecular Neurobiology, Y. Pichon, Ed., Birkhauser Verlag (Basel, Switzerland), pp. 172-209, 1993. Darlison et al., GABAA receptor subtypes: which, where and why?, Seminars Neurosci., 7: 115-126, 1995. Malecot et al, "GABA receptors in the nervous system of an instect, Periplaneta americana", in GABA-B Receptrors in Mammalian Function, N. Bowery et al., Eds, John Wiley and Sons (New York), p. 431, 1990. Chen et al., Cloning and functional expression of a Drosophila gamma-aminobutyric acid receptor, Proc. Natl. Acad. Sci. USA, 91: 6069-6073, Jun. 1994. Ffrench-Constant et al., Molecular cloning and transformation of cyclodiene resistance in Drosophila: An invertebrate gamma-aminobutyric acid subtype A receptor locus, Proc. Natl. Acad. Sci. USA, 88: 7209-7213, Aug. 1991. Lunt, GABA and GABA receptors in inverebrates, Seminars Neurosci., 3(3): 251-258, Jun. 1991. Miyazaki et al., DNA sequence and site of mutation of the GABA receptor of cyclodiene-resistant red flour beetle, Tribolium castaneum, Comp. Boichem. Physiol., 111 B(3): 399-406, 1995. Shotkoski et al., Functional expression of insecticide-resistant GABA receptors from the mosquito Aedes aegypti, Insect Mol. Biol., 3(4): 283-287, 1994. Thompson et al., Cloning and sequencing of the cyclodiene insecticide resistance gene from the yellow fever mosquito aedes aegypti, FEBS, 325(3): 187-190, Jul. 1993. Marullo et al., Expression of human B1 and B2 adrenergic receptors in E. coli as a new tool for ligand screening, Bio/Technology, 7:923-927, Sep. 1989.
Patent History
Patent number: 5767261
Type: Grant
Filed: Nov 8, 1995
Date of Patent: Jun 16, 1998
Assignee: Rhone -Poulenc AG Company (Research Triangle Park, NC)
Inventors: Vincent Wingate (Chapel HIll, NC), Mark Wolff (Cary, NC)
Primary Examiner: Stephen Walsh
Assistant Examiner: Claire M. Kaufman
Law Firm: Scully, Scott, Murphy & Presser
Application Number: 8/554,659